The objective of this project is to investigate the molecular mechanism of DNA replication using a cell free system prepared from bacteriophage T7 infected E. coli. The approach will emphasize studies with purified proteins known from genetic analysis to participate in T7 DNA replication in vivo. T7 DNA polymerase, helix destabilizing protein, gene 4 protein and RNA polymerase have been purified to near homogeneity. The gene 4 protein is a primase which synthesizes short RNA primers for the DNA polymerase on single-strand T7 DNA. However, DNA synthesis on a duplex T7 DNA template requires transcription by the T7 RNA polymerase. We will investigate the mechanism by which transcription stimulates the initiation of DNA replication in vitro. To determine the role of host proteins in phage DNA replication, we have studied the E. coli mutant tsn B, which restricts T7 growth during a late step in DNA replication. This mutant has an altered RNA polymerase which is resistant to inhibition by the T7 gene 2 protein. To determine why inactivation of the bacterial RNA polymerase is required for phase DNA replication, we will study phage mutants which are able to grow on the mutant host.